In Vitro Wound Healing Model: Effects of Chitosan Films Loaded with Gentamicin and Silver Sulfadiazine on the Wound Filling Rate

Advances in Tissue Engineering and Regenerative Medicine

Mini Review Open Access In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on the wound filling rate

Abstract Volume 2 Issue 3 - 2017

Currently, animals use in research experiments has been widely questioned by Animal Maria Gabriela Nogueira Campos,1,3 Henry Protect Institutions and Organizations. Moreover, preliminary tests and scientific 2 3 validation of research hypothesis are required before in vivo tests approval. Thus, Ralph Rawls, Lucia Helena Innocentini Mei, 4 animal testing could take a long time. For all these reasons, the development of an in Neera Satsangi 1 vitro test that could simulate and/or replace animal research would be very interesting. Federal University of Alfenas, Brazil 2 The aim of this work was the development of an in vitro wound healing model for Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, USA evaluation of the healing potential of drug-loaded cross-linked chitosan films. MG- 3Department of Materials Engineering and Bioprocess, State 63, a cell line derived from human osteosarcoma, was used as cell line model. The University of Campinas, Brazil monolayer was cultured and when 100% confluence was reached, a wound was 4Innovative Research Solutions, Inc., USA created using a scraper. The healing process was evaluated for 21 days and cells were stained after 0, 7, 14 and 21 days of incubation. Wounds were treated with chitosan Correspondence: Maria Gabriela Nogueira Campos, Federal film (CH), chitosan-bisulfite blocked diisocyanate cross-linked film (CH-X), chitosan University of Alfenas, Institute of Science and Technology, cross-linked film loaded with gentamicin (CH-X-GE) and chitosan cross-linked film Rodovia José Aurélio Vilela, 11999, Poços de Caldas/MG, Brazil, loaded with silver sulfadiazine (CH-X-SS). The effects of chitosan (CH), bisulfite Tel +55-35-3697-4600, Fax + 55-35-3697-4602, blocked diisocyanate (BBDI), gentamincin (GE) and silver sulfadiazine (SS) on the Email [email protected] wound healing response were microscopically analyzed by the wound filling rate. CH and CH-X films did not show deleterious effects on cell growth compared to the Received: April 27, 2017 | Published: May 16, 2017 positive control (no film) during the studied period. CH-X-GE demonstrated a reduced wound filling rate at the first week due to the burst release of gentamicin at this period. CH-X-SS showed reduced wound filling rates during all period studied probably due to silver cytotoxicity and its accumulation by the cells. Finally, in vitro wound healing rates were compared to in vivo results reported in the Literature. Similar behavior was observed, suggesting that the proposed in vitro wound healing model could potentially replace preliminary in vivo studies.

Keywords: in vitro model, wound healing, wound dressing, chitosan, gentamicin, silver sulfadiazine.

Abbreviations: CH, chitosan film; CH-X, chitosan cross- little suffering to animals as possible, and that animal tests should -linked film; BBDI, bisulfite blocked diisocyanate; GE, gentamicin; only be performed where necessary, respecting the 3R’s principle: SS, silver sulfadiazine; CH-X-GE, chitosan cross-linked film loaded Reducing, Replacement and Refinement of the use of animals in with gentamicin; CH-X-SS, chitosan cross-linked film loaded with research.5 In this context, the development of an in vitro test that could silver sulfadiazine replace preliminary animals testing or serve as pre-screening before in vivo experiments is an interesting, current and crucial research field. Introduction Wound healing is a complex process that involves five phases Animal testing or animal research refers to the use of animals in (hemostasis, inflammation, cellular migration and proliferation, research experiments. Worldwide, about 100 million animals are used protein synthesis and wound contraction, and remodeling), but only annually and either killed during the experiments or subsequently three major phases are effectively considered due to overlap of phases.6 euthanized.1 Animal research is mainly carried out inside universities, Inflammatory, proliferative, and remodeling phases are associated with medical schools and pharmaceutical companies, although some considerable complexity that involves soluble mediators, extracellular commercial facilities provide animal-testing services to industry.2 matrix formation, and parenchymal cell migration.7 Nevertheless, the Groups that supports animal research affirm that it has played a primary goal of wound healing is timely wound closure, which is vital role in every major medical advance and that many major directly related to the wound filling rate.6,8 Additional help might be developments, such as penicillin (mice), organ transplant (dogs), and provide for wound healing in order to accelerate wound closure and/ poliomyelitis vaccine (mice, monkeys) involved animal researches.3 or to improve the regenerated tissue quality. Several approaches on However, the topic is controversial. Opponent groups argue that wound healing treatment are currently available, such as grafts (auto, animal testing is unnecessary, poor scientific practice, poorly allo and xenografts), use of donor keratinocytes, cultured epithelial regulated, that the costs outweigh the benefits, or that animals have an autografts, delivery of growth factors and other molecules, use of stem intrinsic right not to be used for experimentation.4 Nevertheless, most cells and wound dressings.6,7 Wound dressings are most commonly scientists and governments agree that animal testing should cause as used in wounds treatment due to the limitations of grafts (donor site,

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surgical complications, rejection, graft contraction and stability of during the studied period if compared to the positive control (no film), the graft) and autologous cell culture (the time required to culture suggesting that both films are biocompatible and BBDI are non-toxic and prepare sheets of cells for grafting limit its use).6 In addition to MG-63 cells. In fact, no significant difference was observed for the to its general functions, such as covering and protecting the wound wound filling rate of these films, but stimulation on cells growth by against infection, avoiding wound dehydration and allowing wound- CH at day 7 might be suggested. Ueno et al.17 reported that chitosan environment gas/fluid exchanges, an ideal wound dressing should stimulated migration and proliferation of fibroblast and collagen also serve as a matrix for cell adhesion and proliferation, as well as production in a dog wound healing model. Moreover, wound healing a vehicle to delivery growth factors and antibiotics.6–9 There are a experiments using mouse as model have shown that the application number of wound dressing based on polymeric materials available on of chitosan hydrogel onto an open wound induced significant wound market, and chitosan is the most used polymer for this application.6 contraction and accelerated wound healing.18 Xu et al.19 reported the biocompatibility of a genipin-chitosan hydrogel on a rabbit model. Chitosan is a biopolymer derived from chitin, the second most In addition, glycerol phosphate-chitosan hydrogel was assessed in abundant polysaccharides found in nature.10 Due to its interesting experimental osteochondral joint defects in horses and did not cause biological properties such as biocompatibility, biodegradability, relevant clinical effects, inflammatory response or toxic effects in the antibacterial activity and bio-adhesion, it has been widely studied joints.20 Chitosan films were also evaluated on wound repair of horse and applied as a biomaterial in the biomedical area.10,11 Biomedical distal limb and they had not interfered on healing time in the equine applications of chitosan involve implants, scaffolds for tissue model,21 suggesting compatible results of in vivo wound model and engineering, skin substitutes and drug delivery vehicles.12–14 To this reported in vitro wound model. On the other hand, the gentamicin- increase the timeframe and consistency of kinetics of drug delivery loaded crosslinked chitosan film CH-X-GE showed inferior wound from chitosan vehicles, crosslinking might be necessary.15 Several filling rate when compared to positive control in the first week. chemical agents are available for the crosslinking of chitosan: Lin et al.22 reported that application of 2% gentamicin eye drops in glutaraldehyde, genipin, epichlorohydrin, sulfuric acid, etc.; but porcine cornea significantly disturbed the corneal epithelial healing glutaraldehyde is the molecule most commonly used as chitosan rate. Cooper et al.23 investigated the cytotoxic effects of gentamincin cross-linker. On the other hand, there are concerns over the toxicity of on human fibroblasts and keratinocytes, which play an important residual glutaraldehyde, which may compromise the biocompatibility role in wound healing, and found profound effects of gentamicin on of chitosan delivery system.15 A water-soluble bisulfite blocked these cells. Bertolaso et al.24 reported the biochemical mechanism diisocyanate has been recently prepared and used as crosslinking underlying gentamicin cytotoxicity in OC-k3 cells. Although agent for chitosan.16 This bisulfite blocked diisocyanate is devoid of gentamicin ototoxicity and neuphrotoxicity have been extensively any toxic effects of the relative diisocyanate and preferably reacts studied, the dose-dependent deleterious effects of gentamicin have with amine groups of chitosan by urea linkage.15,16 not been well established yet.25,26 Hancock et al.27 studied the retinal Therefore, in this present work, we evaluated the healing potential toxicity of gentamicin in vivo and in vitro and reported reversible of chitosan and chitosan-blocked hexamethylene diisocyanate cross- toxic effects in short-term exposure to gentamicin, while irreversible linked films loaded with gentamicin and silver sulfadiazine on an in retinal damages were observed after prolonged gentamicin treatment, vitro wound healing model. For the in vitro model, human MG-63 suggesting besides dose-dependent, toxicity of gentamicin is also cells, a cell line derived from an osteosarcoma, were seeded in six- time-dependent. After 14 and 21 days of incubation, CH-X-GE film well tissue culture plates (Corning Co, USA). The seeded cells were deleterious effects on wound filling were not observed anymore in our incubated in α-MEM (Minimum Essential Media Alpha Medium) with in vitro wound healing model (CH-X-GE wound filling rate is similar 5% fetal bovine serum (FBS) and humidity atmosphere of 5% CO2 to positive control, CH and CH-X films). This can be attributed to and 95% air at 37˚C. After cells reached confluence, wounds were the decrease on gentamicin concentration in the media, since burst created across the surface of each well (Figure 1A) and the media were release happened in the first week and then minimal amount of replaced. The wounds were then microscopically examined to ensure gentamicin was controlled released by CH-X-GE films during the that cellular and extra-cellular materials were completely removed following two weeks.28 Again, the proposed in vitro wound healing from the wound sites (Figure 1B). At this moment, the chitosan-based model showed analogous results to those found in vivo, suggesting films, previously prepared by solvent evaporation technique and a dose and time-dependent effect of gentamicin on the wound filling sterilized using UV light (Table 1), were placed in each of the wells, rate. Contrary, silver sulfadiazine-loaded crosslinked chitosan film and the positive control was a well with no film. The experiment was CH-X-SS showed reduced wound filling rate during all the analyzed carried out on duplicates. The culture medium was replaced at every period. Silver cytotoxicity id related to its accumulation in the body.29 two days in order to provide essentials nutrients for cellular growth. Thus, the decrease on the amount released by the CH-X-SS trough The wound filling rate was observed for three weeks (21 days). After the weeks does not reduce the cytotoxicity observed at 7, 14 and 21 0, 7, 14 and 21days of incubation, films were removed from the wells days of incubation. Lee et al.30 also reported the cytotoxic effect of and cells were stained with 20ml of T-blue stain for 2 minutes at 37˚C. silver sulfadiazine on HaCaT cells and the delayed epithelialization After staining, all media were removed and cells were washed with in an animal model. In addition, Mi et al.31 observed a marked 1ml of 0.1M phosphate buffer solution (PBS) pH 7.4. Then, PBS was inhibition of 3T3 fibroblasts growth caused by 1% silver sulfadiazine removed and 1 ml of formalin was added. Cells were kept at 4˚C for cream treatment. According to authors, fibroblasts inhibition was 24hours and, after this procedure, formalin was replaced for ethanol significantly reduced by the use of silver sulfadiazine incorporated 70%. At this moment, the wound filling rate was analyzed using a to an asymmetric chitosan membrane.31 Poon et al.32 studied the microscope with photography apparatus. The wound filling rate was cytotoxic effects of silver on keratinocytes and fibroblasts in vitro. qualitatively determined by the amount of cells that grown in each According to their results, silver is highly toxic to both keratinocytes wound site at each analyzed period (Figure 2). According to Figure 2, and fibroblasts, which play an important role in wound healing CH and CH-X films did not show deleterious effects on cell growth process. Moreover, the cytotoxic dosage for skin cells was found to

Citation: Campos MGN, Rawls HR, Mei LH, et al. In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on the wound filling rate.Adv Tissue Eng Regen Med Open Access. 2017;2(3):186‒189. DOI: 10.15406/atroa.2017.02.00031 Copyright: In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on 188 the wound filling rate ©2017 Campos et al.

be similar to that for bacteria, suggesting the use of silver in wound Table 1 Preparation of chitosan-based films for in vitro wound healing treatments should be carefully evaluated since it could delay wound response evaluation. healing instead of accelerate it.32 Several in vivo studies using human and animal burn wounds report the side effects of silver sulfadiazine Sample Film preparation/composition topical treatment,33–35 which corroborate with the results found in our In a 250mL flask, 1.5g of high molecular weight chitosan, in vitro wound healing model. >75% deacetylated (Sigma-Aldrich, USA) was dissolved in 100ml of 1.0% acetic acid solution. After total dissolution, CH the resulted solution was cast in the molds and the films were obtained by solvent evaporation at room temperature.

In a 250mL flask, 0.8g of blocked bisulfite diisocyanate15 was added to 100ml of 1.5% chitosan solution and heated CH-X up to 40˚C under stirring for 24 h. The cross-linked solution was cast in the molds and the films were obtained by solvent evaporation at room temperature.

In a 250mL flask, 0.8g of blocked bisulfite diisocyanate15 was added to 100 ml of 1.5% chitosan solution and heated up to 40˚C under stirring for crosslinking. After 24h, 1.0% CH-X-GE gentamicin sulfate (w/v) was added and the solution was homogenized under stirring for another 24h. Then, the A. Cells being removed by a scraper in order to create a wound solution was cast in the molds and the films were obtained by solvent evaporation at room temperature.

In a 250mL flask, 0.8g of blocked bisulfite diisocyanate15 was added to 100ml of 1.5% chitosan solution and heated up to 40˚C under stirring for crosslinking. After 24h, 5.0% CH-X-SS silver sulfadiazine (w/v) were added and the solution was homogenized under stirring for another 24h. Then, the solution was cast in the molds and the films were obtained by solvent evaporation at room temperature. *All films were sterilized by exposure to UV light for 2

hours each side.

CH, chitosan film; CH-X, chitosan cross-linked film; CH-X-GE, chitosan cross- linked film loaded with gentamicin; CH-X-SS, chitosan cross-linked film loaded with silver sulfadiazine. B. In vitro wound created after cell removal using a scraper (day 0). Cells are stained, while the wound site is not. Conclusion Figure 1 In vitro wound healing model apparatus. In vitro wound healing model results have shown to be consistent to those obtained in animal studies reported in the Literature. Therefore, it could potentially replace preliminary animal testing and/or serve as pre-screening before in vivo experiments, collaborating to the 3R’s principle of reduction, replacement and refinement of animal tests. Acknowledgements The authors thank CAPES and CNPq for financial support. Conflict of interest The author declares no conflict of interest. References 1. The Ethics of research involving animals, Nuffield Council on Bioethi- cs, UK: Springer; 2005. 45 p. 2. National Research Council (US) and Institute of Medicine (US) Com- mittee on the Use of Laboratory Animals in Biomedical and Behavioral Research Use of laboratory animals in biomedical and behavioral research, USA: The National Academies Press; 1998. 3. Animal Research. Foundation for Biomedical Research, USA: Springer; Figure 2 Wound healing results after at 7, 14 and 21 days of incubation. 2017.

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